Heterochromatin is a major component of higher eukaryotic genomes as diverse as humans and fruit flies, and it plays important roles in chromosome and cell biology. Essential functional components are contained within heterochromatic regions, including ribosomal RNA genes, centromeres and telomeres. Alterations in heterochromatin structure and function are important to human health. Errors in chromosome inheritance can result in genomic abnormalities (aneuploidy) associated with a variety of human disorders, including birth defects (e.g. Down's Syndrome), and chromosome rearrangements involving heterochromatic regions are frequently found in cancerous cells. However, major gaps exist in our knowledge of the fundamental nature and overall organization of DNA sequences present in heterochromatin. Molecular and genetic analyses of heterochromatin have been made difficult by the presence of large (up to Megabase-size) arrays of highly-repeated DNAs, or satellites. Our knowledge of the structure and function of higher eukaryotic genomes will remain limited unless we gain a better understanding of the molecular structure of heterochromatin. It is essential that a tractable model system is developed in a higher eukaryote, one that utilizes molecular and genetical approaches to circumvent the unique problems posed by the size and sequence composition of heterochromatin. Without a careful pilot study on a specific, defined heterochromatic region, it is impossible to know if current molecular-genetic approaches can be applied successfully to solving the structure of heterochromatin. This research proposal extends our previous studies into the molecular organization of a 1 Megabase (Mb) heterochromatic region present in the 1.3 Mb Drosophila minichromosome Dp1187. The experiments described here are designed to generate detailed structural information about a specific region of Dp1187 heterochromatin, and to test the efficacy of new experimental approaches to the problems encountered during molecular analyses of regions rich in repeated DNA. The specific aims of this proposal are to make important strides toward completing the structural analysis of Dp1187 centric heterochromatin by l) further investigating the gross organization and composition of the islands of complex DNA and the satellite blocks, 2) cloning a specific region of Dp1187 heterochromatin in large and small insert vector systems, and determining the stability of repeated DNA in such clones, and 3) sequencing a specific region of Dp1187 centric heterochromatin (the complex island Bora Bora) to determine its fine structure. These model system studies will generate information and tools that further our understanding of higher eukaryotic genome structure and provide groundwork for analysis of heterochromatin structure and function in other eukaryotes, including humans.